PROJECTSUMMARY Vascularstiffnessisanindependentpredictorofcardiovascularmortalityindiabetesandrepresentsan understudied,butpotentiallymodifiableriskfactorfordeathinthelargeandgrowingpopulationofAmerican diabetics.However,themolecularmechanismsunderlyingdiabeticvascularstiffnessareonlyrecentlycoming tolight,andtherapeutictargetsremainelusive. Theapelin-APJsignalingaxisisapotentiallytargetablemolecularpathwaythatisdysregulatedin diabetesandactivationofwhichisassociatedwithdecreasedvascularstiffnessinhumansandmice. Importantly,preliminarydatainapelinknockoutmicesuggeststhatthisdecreasedstiffnessmayoccurinpart throughdecreasedvascularmedialfibrosis.MicroRNA-29b(MiR-29b)protectsagainstmedialfibrosisby translationalrepressionofcollagenandelastintranscripts,anditsexpressionisregulatedbycanonicalsecond messengersofapelin-APJbinding.Therefore,wehypothesizethatdiabetesdecreasesapelin/APJ signaling,leadingtoamiR-29b?dependentalterationincollagenandelastinexpressioninthe vascularmediatocauseincreasedvascularstiffness. InSpecificAim1,Iwilluseculturedvascularsmoothmusclecellsexposedtoapelinandhyperglycemia aswellasspecificinhibitorsofcanonicalAPJsignalingtoelucidatethespecificmolecularmechanismsby whichapelinincreasesmiR-29bexpressioninthevascularmedia. InSpecificAim2,Iwilluseamousemodelofdiabetes,theleptinknockout(db/db)mouseaswellas apelinknockoutmicetoassesstheeffectofinvivomodulationofmiR-29bexpressionandapelinondiabetic vascularstiffnessasmeasuredbyexvivopressuremyography.Specifically,IexpecttodemonstratethatmiR- 29badministrationtoapelinKOmicerescuestheirincreaseinvascularstiffness,andthatindb/dbmice,apelin administrationrescuesvascularstiffnessinamiR-29b-dependentmanner. InSpecificAim3,Iwillexaminetheregulationofthesemolecularactorsinhumandiabetesby measuringaorticexpressionofapelin,APJ,miR-29bandmarkersofvascularmedialfibrosisindiabeticvs non-diabeticpatientswhohaveundergonecoronaryarterybypassgrafting.Thiswillconfirmthattheapelin- APJ-miR-29bpathwayisdownregulatedinhumandiabetes,makingitaviabletherapeutictargettoreduce diabeticvascularstiffness. Theproposedexperimentswillidentifymultipleputativetherapeutictargetsandprovideascaffoldon whichtostudymultiplemolecularpathwaysconvergingonvascularstiffness.Asthisrepresentsoneofthefew targetablediseaseentitiesinwhichinterventioncanpreventmortalityandimprovesymptoms,thisprojecthas thepotentialtocontributegreatlytoourtreatmentofdiabeticcardiovasculardisease.